Culture method for long-term maintenance and proliferation subculture of human hepatocytes

Abstract

A method for culturing hepatocytes involves maintenance, proliferation, and passaging of the hepatocytes. The method includes culturing the hepatocytes in a culture medium, which includes a hepatocyte basic medium; CHIR99021, which is a GSK-3 beta inhibitor at a concentration of 0.5-10 μM, SB 431542 or A83-01, which are RGF beta inhibitors, at a concentration of 0.5-10 μM, N-acetyl-cysteine at a concentration of 0.25-25 mM; and Oncostatin M at a concentration of 1-100 ng/mL There is no exogenous gene introduced into the hepatocytes, and the genetic background of the hepatocytes is not changed.

What is claimed is: 
     
         1 . A method for culturing hepatocytes, involving maintenance, proliferation, and passaging thereof, wherein the method comprising:
 culturing hepatocytes by a culture medium,   wherein said culture medium comprises:
 a hepatocyte basic medium; 
 a GSK-3 beta inhibitor at a concentration of 0.5-10 μM, said GSK-3 beta inhibitor is CHIR99021; 
 a TGF beta inhibitor at a concentration of 0.5-10 μM, said TGF beta inhibitor is SB 431542 or A83-01; 
 N-acetyl-cysteine at a concentration of 0.25-25 mM; and 
 Oncostatin M at a concentration of 1-100 ng/mL, 
   wherein there is no exogenous gene being introduced into the hepatocytes, and the genetic background of the hepatocytes is not changed.   
     
     
         2 . The method according to  claim 1 , wherein the method for culturing hepatocytes comprises:
 (1) backing a culture plate for 1 to 24 hours; mixing and suspending primary hepatocytes in an adherent culture; plating; and incubating at 37° C. for 1 to 12 hours;   (2) discarding the adherent culture used in (1) above, transferring the hepatocytes into the culture medium of  claim 1 , and culturing the hepatocytes.   
     
     
         3 . The method according to  claim 1 , wherein the hepatocyte basic medium is supplemented with 10% serum. 
     
     
         4 . The method according to  claim 1 , wherein the concentration of GSK-3 beta inhibitor is 1-4 μM, and the concentration of TGF beta inhibitor is 1-8 μM. 
     
     
         5 . The method according to  claim 1 , wherein the concentration of N-acetyl-cysteine is 0.5-12.5 mM. 
     
     
         6 . The method according to  claim 1 , wherein the concentration of Oncostatin M is 5-80 ng/mL. 
     
     
         7 . The method according to  claim 1 , wherein the culture medium further comprises an ingredient selected from the group consisting of:
 human epidermal growth factor at a concentration of 5-100 ng/ml;   human fibroblast growth factor at a concentration of 5-100 ng/ml;   human hepatocyte growth factor at a concentration of 5-100 ng/ml;   dexamethasone at a concentration of 0.05-1 μM;   platelet derived factor at a concentration of 1-100 ng/ml; and   triiodothyronine at a concentration of 1-100 ng/ml.   
     
     
         8 . The method according to  claim 1 , wherein the culture medium further comprises sodium pyruvate. 
     
     
         9 . The method according to  claim 1 , wherein the hepatocytes are human hepatocytes.